Eukaryotic messenger RNA (mRNA) contains three regions, 5′ untranslated region (5′UTR), protein coding region, and 3′ untranslated region (3′UTR). The 3′UTR is the sequence towards the 3′ end of the mRNA and contains a polyadenylation signal and a polyadenylation site. The 3′UTR is an important regulatory element in many instances it dictates the mRNA stability and it can also regulate translation efficiency. For example, AU-rich elements tend to destabilize mRNAs (Dalphin et al. 1999). The mRNAs can be classified into three categories based on their half-life: (a) unstable mRNAs with a short half life, for example, less than 2 hours, and (b) intermediately stable mRNAs with a half life that exceeds, for example, 2 hours, and (c) stable mRNAs with a half life that exceeds 8 hours. Many of the housekeeping genes tend to code for stable mRNAs, such as but not limited to globin mRNAs (Chen and Shyu 1994; Shaw and Kamen 1986), β-actin, ribosomal proteins, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
Expression vectors are important research and biotechnology tools. In research they are used to study protein function while in industry they are used to produce (therapeutic) proteins. Optimization of expression vectors for efficient protein production has been sought and practiced in many ways, mostly by optimizing strong promoters that lead to an efficient transcription of the mRNA encoded by the protein coding region in the expression vector. Examples of these are the CMV immediate early promoter, SV40 promoter, elongation factor (EF) promoter, and chicken β-actin promoter (Foecking and Hofstetter 1986; Kobayashi et al. 1997).
Likewise, strong polyA signal cassettes were also used to augment the protein expression by providing strong polyadenylation signals that stabilize mRNAs which are produced from the expression vectors. Among the widely used polyA signals are the bovine growth hormone (bGH) polyA signal (U.S. Pat. No. 5,122,458 by Post et al.: Use of a bGH GNA polyadenylation signal in expression of non-bGH polypeptides in higher eukaryotic cells) and SV40 polyA signal (Goodwin and Rottman 1992). Unlike termination signals in bacteria, where the 3′ end of the mRNA is formed by the NA polymerase that is simply dropping off the DNA and ceasing transcription, in eukaryotes cleavage occurs that is followed by binding of the polyadenylation complex to an AAUAAA sequence near the end of the mRNA. This complex contains an endonuclease that cuts the RNA about 14-30 bases downstream of the AAUAAA sequence and a polymerase that adds a string of polyA forming the polyA tail (Wigley et al. 1990).
Since the 3′ UTR and polyA signal context sequence may influence nuclease degradation of plasmid DNA vectors particularly after delivery and during trafficking to the nucleus, a novel approach that circumvents this problem is described. In this invention, an approach to further optimize expression of proteins is described which uses a hybrid 3′UTR downstream of the protein coding region that consists of two regions, wherein one region is from an 3′UTR of a stable mRNA and the other region is a poly A signal containing region from an 3′UTR of another mRNA.